Our genetic information is contained on DNA and the proper organization of this DNA within the nucleus is important for our development and individual cell health. Disorder of DNA within the nucleus can lead to aberrant expression of genes critical to cell cycle regulation and ultimately can facilitate cell metastasis. This organization requires varying degrees of packaging beginning with the nucleosome core. Multiple cores are then further condensed to form chromatin fibers. The proper organization of the chromatin fibers within the nucleus is essential for eukaryotic gene expression but the mechanism for packaging the chromatin within the nucleus is not well understood. Data suggests the organization and compartmentalization of chromatin fibers into higher-order chromatin domains can involve insulator elements. These insulator elements and their binding proteins have been shown to cluster in the nucleus to form insulator bodies. Clustering of insulator elements into insulator bodies can be a means of organizing chromatin. In this way, insulators may provide a strategy by which cells control the establishment and maintenance of independent transcriptional domains. Organizing the chromatin into loops may be a way of making certain DNA sequences more accessible to transcriptional machinery and in essence dictating which genes can be turned on and off. This proposal seeks to better define the mechanism by which insulator components regulate higher-order chromatin organization. To this end, I will conduct a detailed analysis of one newly identified protein that regulates insulator complexes as well as identify additional proteins involved in insulator body formation. I have identified Topo II as a potent regulator of insulator complexes and higher-order chromatin structure. Topo II will be characterized and its genetic and biochemical basis for interaction with other insulator components will be assayed using gel-shift, co-IPs, and yeast two-hybrids. In addition, the enzymatic and scaffolding functions of Topo II will be individually tested for their role in insulator body formation by inhibiting each function in separate assays. Finally, an RNAi cell based screen will be conducted to identify novel proteins involved in insulator body formation and regulation. From these studies I hope to gain insight as to the precise mechanism by which insulators and accessory proteins establish and regulate higher-order chromatin domains and how these domains affect transcriptional control and cell function.